Abstract Genome instability, i.e., the acquisition of gene mutations and both structural and numerical chromosomal aberrations, is a hallmark of cancer. Genome instability, however, also significantly contributes to tissue degeneration and organ dysfunction in aging. How age-related changes in genome maintenance contributes to cancer risk remains unknown. The main goal of this application is to determine the molecular genetic basis of this association. Studying normative aging in the mouse Dr. Montagna (extramural PI) established a significant accumulation of aneuploidy in non-neuronal cells (NeuN-) isolated from the cerebral cortex, but not the cerebellum, of old mice. In this application we propose to build on this seminal observation and join forces with Dr. Ried (intramural co-I), an expert in cancer genetics, to determine how changes in genome stability at old age contribute to increase cancer susceptibility. To do so we propose to employ an arsenal of complementary, cutting-edge experimental approaches used in our labs. Multicolor interphase FISH (miFISH) to measure aneuploidy and copy number changes, and single-cell whole genome analyses to simultaneously detect all forms of large scale genomic instability. Both assays are amenable to deal with the substantial, cell-to-cell stochastic variation known to be associated with aging. Here we will test the novel hypothesis that astrocytes of the cerebral cortex, but not the cerebellum, accumulate genomic instability during aging and this ultimately increase their risk for transformation. The risk to develop brain tumors increase with age and most adult brain tumors develop in the cerebral cortex, unlike in children where tumors originate in the cerebellum. Our hypothesis will be tested along two specific aims. In Aim 1 we will determine the genetic landscape of astrocytes isolated from the cortex and the cerebellum during normative aging to establish mutation patterns in aging brain cells. In Aim 1 we will establish the genetic landscape of non-tumor astrocytes isolated from GBM patients relative to patient matched tumor cells. All experiments will utilize cortical and cerebral brain biopsies obtained from post mortem donors from the NIH NeuroBioBank. A major strength of this application is the research team. Dr. Montagna and Dr. Ried have known each other since 1998, and they have 18 joint publications. Dr. Montagna will contribute her expertise in chromosome instability and aging, and Dr. Ried will provide knowledge on cancer genomics.